DE60127175T2 - STORAGE-STABLE POWDER COMPOSITIONS WITH INTERLEUKIN-4 RECEPTOR - Google Patents

Abstract

The present invention provides storage stable dry powder compositions of IL-4R. The powder compositions demonstrate superior chemical and physical stability over their solution counterparts, particularly upon storage under varying conditions of temperature and humidity. Moreover, the powders, as prepared, possess good aerosol properties, which are maintained upon storage.

Description

The The present invention relates generally to spray-dried, inhalable Interleukin-4 receptor (IL-4R) powder compositions and methods for the manufacture and pulmonary administration of such compositions. The powders according to the invention are particularly stable in terms of monomer content and aggregate content, both in production and storage, and possess additionally excellent aerosol properties, even in the absence of stabilizing carriers or excipients. The powders of the invention are, when applied in deep lung areas, suitable for the treatment of allergic Diseases such as Asthma, atopy and atopic dermatitis.

Interleukin 4 (IL-4, also known as B-cell stimulating factor or BSF-1) is a cytokine produced by T helper cells, mast cells and basophils. IL-4 has been shown to possess a broad spectrum of biological effects including co-stimulation of the growth of T cells, mast cells, granulocytes, megakaryocytes and erythrocytes. In addition, IL-4 stimulates the proliferation of multiple IL-2 and IL-3 dependent cell lines, triggers the expression of class II major histocompatibility complex molecules in resting B cells, influences the formation of IgE, and enhances the secretion of IgE and IgG ₁ isotypes by lipopolysaccharide-stimulated B cells. IL-4 has been shown to play a crucial role in the development of allergic diseases and is very often associated with asthma and allergies; or with diseases characterized by respiratory distress.

IL-4 binds to the IL-4 receptor (IL-4R), an endogenous membrane-bound Protein on the surface certain cells. Upon such binding, IL-4R transduces a biological signal to various immune effector cells and This triggers a cascade of events that are too clinical Symptoms (Renz, H. et al., 1991, J. Immunol., 146 (9): 3049-55). Nucleotide and protein sequence determination for IL-4R is executed Service. Fully developed, human IL-4R has three domain structures on: an extracellular domain (about 207 amino acids), a membrane passage region (about 24 amino acids) and an intracytoplasmic domain (about 569 amino acids) (European Patent No. EP 585-681 (1994)). soluble IL-4R (sIL-4R) is also isolated, cloned and extensively researched been (European Patent No. EP 367-566 (1997); Mosley et al., 1989, Cell, 59-335, 1989; U.S. Patent Nos. 5,767,065 and Garrone, P. et al., 1991, Eur. J. Immunol., 21 (6): 1365-9). IL-4 preferentially binds to sIL-4R in solution rather than at the endogenous cell surface IL-4R and thereby prevents cellular activation and blocks the biological response, e.g. the cascade of effects with IL-4 and its binding to the endogenous receptor (Renz, H. et al., 1991, supra, and Renz, H., 1999, Inflamm. Res., 48 (8): 425-31).

IL-4R is as an immunosuppressant and an anti-inflammatory drug and the administration of IL-4R can be described in the Treatment of conditions such as Allergy, rhinitis, atopic dermatitis, Rheumatoid arthritis, graft rejection, chronic graft-versus-host reaction (GvH) and systemic lupus erythematosus (SLE), be beneficial (See, e.g., U.S. Pat No. 5,856,296; Renz, H. et al., 1992, J. Invest. Dermatol., 99 (4): 403-8; Hackstein, H. et al., 1999, Tissue Antigens, 54 (5): 47-17; Rivas, D., et al., 1995, J. Autoimmun., 8 (4): 587-600 and Schorlemmer, H.U. et al., 1995, Inflamm. Res., 44, Erg. 2: S 194-6).

Like many biopeptides, IL-4R tends to be unstable. It tends to decompose and / or aggregate under extreme conditions (eg, strongly acidic or basic pH, high temperatures) and is susceptible to oxidants and endogenous proteases. The inherent chemical and physical instability of IL-4R makes the pharmaceutical formulation particularly problematic. In order to maintain the stability and bioactivity of the protein, common IL-4R formulations are primarily solution-based and are stored as lyophilisates prior to administration (eg, US Pat. Nos. 5,856,296, 5,767,065 and 6,063,371). A soluble solution-based IL-4R peptide composition for administration by inhalation, Nuvance ^™ , is currently being clinically tested for the treatment of asthma (Borish, LC et al., 1999, Am. J. Resp. Crit. Care Med. 6): 1816-23).

Solution-based formulations of IL-4R suffer from other disadvantages than those associated with solution-phase instability are different. First, solution-based formulations take up more space and require more effort than solid formulations and are therefore more expensive. Moreover, they generally need to be refrigerated (typically stored in an environment of 2 to 8 ° C), which further restricts the storage and transportation options. In addition, many solution-based formulations over time show protein concentration loss presumably due to the formation of dimers and other protein aggregates in solution. Such formulations often have to be supplemented with stabilizing additives, such as buffer substances and / or antioxidants, in order to obtain the sol minimize instability. Accordingly, it would be desirable to provide a solid or powder-based composition of IL-4R, particularly one that could not only be stably prepared and stored, but additionally administered in solid form, such as an inhalable dry powder. Many preclinical and clinical studies with inhaled proteins, peptides, DNA and small molecules have demonstrated efficacy both within the lungs and systemically.

powder formulations represent an alternative to solution formulations and proteins, when desired in powder form, most frequently prepared as lyophilizates (e.g., U.S. Patent No. 5,856,296). Unfortunately Freeze-dried powders are typically used as a powder cake shaped, what additional Abrasive and grinding and possibly sieve processing steps require flowing To provide powder. In recent years, spray drying has been used as an alternative method of making a Series of protein-based therapeutic powders, in particular for aerosolized administration (e.g., International Patent Publications Nos. WO 96/32149; WO 95/31479; WO 97/41833, Inhale Therapeutic Systems, Inc. assigned). Unfortunately, certain proteins, and cytokines, tend to be in particular, to decomposition during spray-drying and the loss of their secondary structure (Maa, Y.F., et al., J. Pharm. Sciences, 87 (2), 152-159 (1998)). For a representative Cytokine, human growth hormone, Mumenthaler reported that spray-drying at 90 ° C to form 4% insoluble Aggregates and led to the formation of 21% soluble aggregates - a loss of 25% intact protein (Pharmaceutical Res., 11, 12-20 (1994)). The instability of the illustrative cytokine, hGH, was continued by Maa, Y.F., et al., Ibid, 42% aggregate formation (soluble and insoluble) with atomization a solution reported by hGH.

Additionally owns sIL-4R a number of potential instability sites, resulting in both solution-based instability as well as instability in the solid state leads. Specially contains sIL-4R 7 cysteines (Cys11, 21, 31, 51, 61, 63 and 184), which is at least a free sulfhydryl guaranteed, which for intermolecular disulfide bonds available can be. Such intermolecular disulfide bonds lead to the easy formation of dimers, trimers and other aggregates with itself. Thus, this molecule is particularly prone to instability. Next aggregation-prone In places, the IL-4R peptide also exhibits sites susceptible to decomposition on. For example, close the sites that are likely to be oxidatively vulnerable, four methionine residues (Met3, 16, 25 and 67). additionally gets at the amino acid residues 145-146 an acid labile Asp-Pro bond found that is cleavable at low pH. Two likely deamidation sites include Asn-Gly (26-27) and Asn-Gly (56-57), although the molecule has numerous possesses other potential deamidation residues (Asn and Gln).

Consequently The inventors did not face the difficult task to provide only an improved dry powder formulation with IL-4R, around some of the solution-based ones Formulations associated with IL-4R disadvantages, as described above, to overcome, but also the factors affecting instability and aerosol properties influence of IL-4R, balance to provide a stable dry powder formulation, the for pulmonary administration is suitable to arrive. That is, before The present invention was the development of a chemical and Physically stable, bioactive dry powder with IL-4R, the also for the aerosolization of necessary physical properties (e.g. high dispersibilities, which remain stable over time, suitable aerodynamic size), unknown.

The The present invention is based on the unexpected discovery of chemically and physically stable spray dried powder compositions of IL-4R, although known from such molecules (i.e., cytokines) is that they are particularly unstable when exposed to shear stress, the fluid-wall interactions, the high temperature conditions and the like of spray-drying get abandoned. Surprised shows the spray-dried according to the invention Powder both a monomer content and an aggregate content, the essentially unchanged are opposite those of the solution before spray drying. moreover the invention provides IL-4R dry powder compositions ready, which are storage stable, both monomer content and Concerning aggregate content, even under extreme humidity conditions. This means, the spray-dried ones described here Powders show both excellent chemical and physical Stability, as well as having good dispersibilities (i.e., aerosol properties), what she for Makes it suitable for use on the lungs.

In one embodiment, the invention provides a spray-dried IL-4R powder composition which can be stored for extended periods of time, 14 days or more, under extreme humidity and temperature conditions without substantial changes in aerosol performance, chemical and / or physical property To experience bioactivity and the like. More specifically, the IL-4R content of the powder compositions of the present invention is substantially unchanged the suspensions or solutions prior to spray-drying, ie it undergoes minimal aggregate formation and / or minimal protein monomer loss over time.

The IL-4R powder composition which is negligible in production and storage Decomposition shows, in the absence of stabilizing additives or excipients can be prepared or may further be a pharmaceutical acceptable Contain excipients. Preferred excipients include zinc salts, Citrate, leucine, and combinations thereof.

The IL-4R powder composition preferably has a monomer content on, essentially unchanged is opposite that of the solution or suspension before drying. The change in monomer content is here expressed as a percentage decrease (compared to the solution or suspension before drying) shown. The decrease of the monomer content is preferably less than about 10%, stronger preferably less than 7%, most preferably less than 5%.

The IL-4R powder composition preferably exhibits minimal aggregate formation across from the solution or suspension before drying. The proportion of aggregate formation is here as a percentage increase (compared to the solution or suspension before drying) shown. The increase in the aggregate content is preferable less than 10%, stronger preferably less than 7%, most preferably less than 5%.

Additionally include the IL-4R powder compositions of the invention Particles that effectively penetrate into the alveoli, that is, the in a special embodiment have a mass median diameter (MMD) of less than about 10 μm, preferably less than about 7.5 microns, and most preferably less than 5 μm In diameter. In a particularly preferred embodiment For example, the powder is composed of particles having an MMD of about 1.0 to 3.5 μm.

Further embodiments the IL-4R powder compositions of the invention include spray-dried IL-4R particles that have a mass median aerodynamic diameter (MMAD) of less than about 10 microns, preferably less than about 5.0 microns, and more preferably less than about 3.5 microns. In a particularly preferred embodiment the MMAD is in the range of 1.5 to 3.5 microns.

Also Included in the invention is an aerosolized IL-4R powder formulation and an IL-4R powder in a unit dose dosage form.

In another embodiment the invention is directed to a method of administering an IL-4R powder composition, as described here, to the lungs of a patient whose needs, directed. In the method, a composition, as described above, by inhalation in aerosolized Administered form.

The Invention also, in yet another embodiment, comprises a process for preparing a dispersible, dry IL-4R powder composition, having the features described above.

In an embodiment For example, the respirable IL-4R powder composition is prepared by combining the IL-4R drug (s) in a suitable solvent, to a mixture or a solution to form and spray-dry the mixture or solution, to obtain individual, substantially amorphous particles, preferably in the form of a dry powder. The IL-4R stays on spray drying essentially intact, resulting in powder particles in which the extent of protein decomposition (as indicated by decrease in monomer content and aggregate formation) is irrelevant.

One optional pharmaceutical excipient may further be added to the solvent be added to a homogeneous solution or a heterogeneous mixture to form, so that spray drying the solution or the mixture provides particles which, together with IL-4R, a Excipients, a buffer and any other ingredients that in the solution or the mixture is present. In another embodiment For example, the pharmaceutical excipient may be separately dissolved and spray-dried to provide separate, but in addition to give administrable powder particles.

These and other objects and features of the invention will become more apparent if the following detailed description along with the attached Illustrations and examples will be read.

1 This figure represents a graph of the effect of temperature on the monomer content of the spray-dried powders, Formulations 1 (A) -1 (D) of Example 1 (2-week stability data).

2 This figure presents a graph of the relative humidity effect on the monomer content of the spray-dried powder, Formulations 1 (A) -1 (D) of Example 1 (2-week temperature stability data).

3 This figure represents a graph of the effect of temperature on the monomer content of the spray-dried powders, Formulations 1 (A) -1 (D) of Example 1 (2-week temperature stability data).

A. Definitions

The The following terms as used herein have the meanings indicated.

in the Context of the present invention, "IL-4R" and "sIL-4R" refer to the extracellular domain of the cell-bound protein, that as a receptor for the cytokine, interleukin-4, is used. As discussed below IL-4R as used herein does not indicate a single peptide sequence limited, but is meant to encapsulate any known protein that has IL-4R activity, including from natural and synthetic sources of IL-4R, as well as agonists and Analogs thereof, as far as those associated with the native peptide therapeutic activity preserve.

As Used here, the term "agonist" refers to compounds that have the effect of native Imitate connection. An agonist may be a peptidic or a non-peptidic Be connected.

As As used herein, the term "analog" refers to those compounds in which one or more amino acids substituted, removed (i.e., fragments), added or otherwise modified from the native (wild type), human sequence and at least about 10, 20, 30 or 40% and preferably at least 50%, 60% or 70% and most preferably at least 80%, 90%, 95%, 100% or more than 100% bioactivity of the of the native (non-synthetic), endogenous peptide. The receptor specificity is possibly substantially similar to the native (wild-type), endogenous peptide. Typically, the receptor affinity will be at least 30%, 40% or 50% of that of the native (wild-type), endogenous peptide be; stronger preferably at least 60%, 70%, 80%, 90%, 95%, 100% or more than 100%.

Compositions according to the invention are considered "inhalable" if for inhalation therapy (i.e., capable of being delivered through the mouth or the Inhaled and pulled into the lungs) and / or pulmonary Delivery (i.e., local delivery to the tissues of the deep lung area and absorption by the epithelial cells therein into the bloodstream) are suitable. Compositions according to the invention are preferably for rapid systemic absorption by the Lungs, i. with maximum values in the blood in less than 60 minutes, suitable.

As used here, the "deep lung area" refers to the alveolar areas the lung (as opposed to the bronchial areas). A composition suitable for "inhalation therapy" is a composition readily soluble in aerosolization (i) an oral inhalation or intranasal delivery device and (ii) from a mammalian individual either can be inhaled through the mouth or nose, so at least a proportion of the particles through the mucous membranes of the nasal passages or the Lung is resobed. One for the "pulmonary Administration "suitable Composition comprises particles, at least one of which, at delivery over Inhalation in aerosolized form that reach tissues of the lungs, including of the deep lung area.

"Orally inhalable" compositions are those inhalable compositions that are particularly the oral inhalation are adjusted. Likewise, "nasal inhalable "compositions of those inhalable compositions particularly sensitive to nasal inhalation, i.e. intranasal delivery to the upper respiratory tract.

"Dry powder" refers to an inhalable composition containing finely dispersed solid particles that are relatively free flowing and capable of (i) being readily dispersed in an inhaler and (iii) inhaled by a subject such that a proportion of the particles reach the lungs To allow penetration into the alveoli The dry powder may be crystalline, an amorphous glass or a mixture from both forms. A dry powder typically contains less than about 10% moisture, preferably less than about 3% moisture, and more preferably contains less than about 3% moisture.

"Delivered Dose "or" ED " dose) provides an indication of the delivery of a drug formulation from a suitable inhaler after a launcher or dispersion event. More specifically, for dry powder formulations, the ED a measure of Percentage of powder withdrawn from a unit dose pack and the mouthpiece an inhaler leaves. The ED is defined as the ratio that of an inhaler delivered dose to the nominal dose (i.e., the mass of the powder per unit dose placed in a suitable inhaler before firing). The ED is an experimentally determined parameter and typically becomes with an in vitro device construction, which determines the delivery of a dose to the patient. To determine an ED value, a nominal dose of dry powder, typically in unit dosage form, in a suitable dry powder inhaler (as in U.S. Patent No. 5,785,049, Inhale Therapeutic Systems assigned, described), which is then actuated, whereby the powder is dispersed. The resulting aerosol cloud is then with vacuum from the device pulled where it on a, attached to the device mouthpiece tared filter is caught. The amount of powder that reaches the filter forms the delivered dose. For example, for a 5 mg, dry powder-containing Dosage form, which is placed in an inhalation device, if the Dispersion of the powder for the recovery of 4 mg of powder on a tared filter as described above, the delivered dose for the dry powder composition: 4 mg (delivered dose) / 5 mg (nominal dose) × 100 = 80%. For non-homogeneous Powder, ED values provide an indication of drug delivery from an inhaler after firing instead of the dry powder and are based on the amount of drug based instead of the total weight of the powder. Corresponds accordingly for MDI and nebuliser dosage forms the ED with the percentage of drug coming from a single dose Dosage form is drawn and leaves the mouthpiece of an inhaler.

A "dispersible" powder is one, which has an ED value of at least about 30%, preferably at least about 40%, stronger preferably at least about 50%, and even more preferably at least about 55%.

"Mass Median Diameter "or" MMD "is a measure of the mean Particle size, since the powder according to the invention generally polydispersed (i.e., consisting of a range of particle sizes). MMD values as reported here, are determined with a centrifuge settling process, although any number usually used techniques for measuring the average particle size (e.g., electron microscopy, light scattering, laser diffraction.

"Mass Median aerodynamic diameter "or" MMAD "is a measure of the aerodynamic size of a dispersed particle. The aerodynamic diameter is used to an aerosolized powder in terms of its settling behavior and is the diameter of a unit density sphere, which has the same settling velocity in air as the particle. The aerodynamic diameter encloses particle shape, density and physical size of a Particle. As used herein MMAD means the mean or Median of the aerodynamic particle size distribution of an aerosolized Powder determined with cascade impaction, if not stated otherwise.

The " _{Fine Particle} Fraction" (FPF _<3.3μm ) is defined as the dose of powder that is below 3.3 microns, as determined by cascade impaction, which corresponds to the total mass below Step 3 of an Andersen impactor. when operating at a flow rate of 1 cfm (28.3 L / min).

"Pharmaceutically compatible Excipient "and" pharmaceutical compatible Carriers "are synonymous and denote excipients contained in the formulations of the invention be taken into the lungs together with the particles can, without significant toxicological side effects for the individual and in particular for the lungs of the individual.

"Pharmacologically effective amount" or "physiologically effective amount" is the amount of IL-4R present in a composition of the invention as described herein which is required to maintain a desired level of IL-4R in the bloodstream of the tissue to be treated To provide individuals and thereby produce an expected physiological response when such a composition is administered by inhalation for deposition in and absorption from the lungs. The exact amount will depend on many factors, eg the particular Il-4R used (eg natural or synthetic, full size or a fragment or analog), the dispenser used, the physical egg properties of the powder, the intended use by the patient (eg, the number of doses administered per day), and factors related to the patient (eg, age, height, weight, health, etc.), and may be determined by a person skilled in the art Information provided here.

A "surface active Means "is a Excipient, the surface activity (e.g. with surface tensiometry measured), as characterized by its ability to surface tension the liquid, in which he solved is to decrease and the rapid spread of its associated Drugs on mucosal surfaces too cause. Surface tension, the one with the interface between a liquid and another phase is the property of one Liquid, due to which the surface molecules have a show inward attraction. The term also closes Detergents, emulsifiers, penetration improvers and wetting agents one.

With "water-soluble Peptide "is a Peptide meant having a solubility in water of at least 0.5 mg / ml, and more preferred of at least 1 mg / ml.

"Amino Acid" means any A compound containing both an amino group and a carboxylic acid group contains and close pharmaceutically acceptable Salts thereof. Although the amino group is most commonly attached to the carboxy function occurs adjacent position, the amino group to any Place within the molecule be positioned. The amino acid can also be extra contain functional groups, e.g. Amino, thio, carboxyl, Carbamide, imidazole etc. The amino acids can be synthetic or natural and can either in their racemic or optically active (D or L) forms used, for example as a single optically active Enantiomer or as any combination or ratio of Enantiomers.

A "dispersant" refers to one Component of the respirable IL-4R powder compositions described herein, when in 0.01 to 99% by weight of the composition is present, preferably from 0.01 to 70 weight percent, effective is the dispersibility of the inhalable IL-4R powder compositions (determined by determining the delivered dose) by at least 10%, compared with the dispersibility of the inhalable IL-4R composition without the dispersant.

"Pulmonary Lung bioavailability "or" relative Bioavailability "is the percentage the administered IL-4R dose that had been deposited in the lungs and that is resorbed and becomes available in the systemic circulation of a mammal, across from the percentage resulting from an intramuscular or subcutaneous injection site be resuspended in the blood. Representative model systems for Determination of lung bioavailabilities shut down Rat, rabbit and monkey. Pulmonary lung bioavailabilities can on direct intratracheal administration or administration Inhalation of an inhalable IL-4R powder composition, as described here.

"Bulk density" refers to the density of a powder before compaction (i.e., the density of a non-compressed Powder) and is typically using a known USP method measured.

"Essentially unchanged ", as in relation to Monomer content or aggregate content of an IL-4R powder composition according to the invention refers to a composition that is a change not more than about 2% in either the monomer content or Aggregate share shows when compared with those of the corresponding solution or suspension before spray drying.

A "minimal Increase " when used in relation to the IL-4R aggregate content in a spray dried IL-4R powder an increase in the proportion of aggregates of no more than about 10% compared to the proportion of aggregates in the corresponding solution or suspension before spray drying.

A "minimal Change "when used in terms of IL-4R monomer content in a spray-dried IL-4R powder a change (i.e., decrease) of the monomer content of not more than about 10% in Comparison to the proportion of IL-4R monomer in the corresponding solution or Suspension before spray drying.

"humidity Condition " an environment with a relative humidity greater than 30% relative humidity (RF). A particularly humid environment is an environment that has a relative humidity of greater than about 60% RF, with high humidities in the range of about 70% to 75% RH or greater.

B. Components of inhalable IL-4R powder composition

The The present invention provides highly dispersible, respirable powder compositions ready containing IL-4R for pulmonary delivery. The ones described here Overcome powder compositions Many of the problems encountered so far when administering peptidic Drugs on systemic paths often encountered, especially the with solution-based Formulations associated with IL-4R problems. Examples of such Close problems extended Response time (e.g., the time between administration and onset of physiological response), low systemic absorption and relatively low Concentrations in tissues and secretions, the inability maintain acceptable serum levels, and the instability of the peptides, and cytokines in particular, in solution one.

The Compositions of the invention are particularly effective for the treatment of allergic diseases and suffering, such as Asthma and atopic dermatitis. moreover are the compositions described herein which are spray dried IL-4R powder included, surprisingly stable (i.e., show minimal chemical and physical decomposition in production and storage, even under extreme temperature and Humidity conditions). That is, the ones provided here Powders are surprisingly robust, even in the absence of stabilizing or dispersibility-increasing excipients. The IL-4R powders according to the invention (i) be used with aerosol dispensers slightly dispersed (i.e., show good aerosol performance), (ii) show surprisingly good physical and chemical stability during powder production and processing and storage and (iii) become reproducible prepared (Examples 1-5).

The inhalable, according to the invention IL-4R powder compositions contain IL-4R and optionally, but not necessarily, a pharmaceutically acceptable excipient. The parts the inhalable according to the invention IL-4R powder compositions will now be described.

IL-4R for use in the invention is generally characterized as follows. Endogenous, fully developed interleukin-4 receptor is considered to be a 140 kDa membrane glycoprotein that binds IL-4 with high affinity, (Idzerda, R. L. et al., 1990, J. Exp. Med., 171 (3), 861-873; Jacobs, C.A. et al., 1991, Blood, 77 (11): 2396-2403). The extracellular domain of the human IL-4R, cloned and formed in CHO cells in serum-containing media, is a highly glycosylated (N-linked) and sialylated protein, this is a non-glycosylated one Molecular weight of 23.9 kDa and 209 amino acid residues contains. The extracellular Domain IL-4R is between residues 24 and 234 of the mature interleukin-4 receptor localized. Mass spectrometric data show that the molecular weight of the protein is about 37 kDa, which suggests at least 35% glycosylation. In SDS-PAGE analysis elutes the protein as a 54 kDa band. The pI of IL-4R is 3.36 to 5.18 as determined with isoelectric focusing. The unfolding transition temperature, as determined by DSC 57.8 ° C and the process of unfolding is highly reversible.

IL-4R for use in the compositions described herein can be purchased from a commercial source or can be recombinant prepared, for example, by a method described in U.S. Pat. No. 5,767,065 and to Armitage et al. in Adv. Exp. Med. Biol. 1991; 292: 121-30. The IL-4R may be neutral (i.e. uncharged) or in the form of a pharmaceutically acceptable Salt, for example an acid addition salt, e.g. an acetate, maleate, tartrate, methanesulfonate, benzenesulfonate, toluenesulfonate, etc., or a salt with an inorganic acid, e.g. a hydrochloride, hydrobromide, sulphate, Phosphate, etc. Cation salts can also be used, such. Salts with sodium, potassium, calcium, Magnesium, or ammonium salts.

The Amount of contained within the respirable powder compositions IL-4R will be the amount necessary to be a therapeutic effective amount (i.e., amount required to treat the therapeutic Effect) of IL-4R per unit dose during the course of a daily Dispensing dosing pulmonary. In practice this will vary depending on the special IL-4R (e.g., natural vs. synthetic, full size vs. Fragment, and its corresponding bioactivity), the patient population and the delivery requirements. Due to the highly dispersible Nature of the inhalable invention Powder minimizes losses in the inhaler, which means that actually more of the powder dose is delivered to the patient. That in turn correlates with a lower required dosage to that desired to achieve therapeutic goal.

Generally, the total amount of IL-4R contained in the respirable powder compositions will range from 1 to 100% of the total weight of the respirable powder composition, preferably from 5 to 98%, more preferably from 10 to 95%, even more preferably from about 45 to From 95% to about 50% to about 90%. A preferred dry powder composition is from about 40% Contains 80% IL-4R (wt .-% of the composition) and is more preferably from about 0.2 to 99 wt .-% IL-4R included.

The required effective amount of IL-4R is from a patient to the next vary and from one regimen to the next. The amount and frequency the administration becomes natural of such factors as the nature and severity of the treated Indication, the desired Response, the patient population, the patient's condition and depend so forth.

The commonplace Dosage for parenterally administered sIL-4R to elicit a biological effect is in the range of about 1 ng / kg / day to about 10 mg / kg / day, stronger preferably about 5 μg / kg / day to about 2 mg / kg / day. Such parenteral formulations with IL-4R are described in U.S. Pat. Nos. 5,856,296 and 6,063,371, supra discussed, discussed. However, since pulmonary delivery is often more efficient is as a parenteral delivery, can the required dosages vary, and may actually be slightly lower be used as those in parenteral formulations. The gift of IL-4R, in particular for the treatment of allergic diseases, such as. Asthma typically occurs weekly. The weekly Dosage of an IL-4R powder by inhalation may range from about 0.1 to 10 mg, stronger preferably between 0.5-5 mg, even stronger preferably between 1-2 mg. Accurate dosages are governed by various factors, e.g. the concentration of IL-4R in the spray-dried powder. Desired dosages are typically achieved in 1 to 10 breaths or 2 to 6 breaths, more preferably in 1-4 Breaths depending on the exact unit dosage form used.

The Efficiency of systemic IL-4R delivery via the method described herein; i.e. the percentage of administered dose affecting the bloodstream achieved (e.g., pulmonary pulmonary bioavailability) from a solid Inhalation dosage form will typically be at least about 1%, stronger preferably at least about 2%, typically at least about 3-5%. In one stronger preferred embodiment is the efficiency systemic delivery into the bloodstream from the lungs at least about 15% to 30%.

C. Excipients and additives

The inhalable, according to the invention Powder compositions may be "neat", i.e. without pharmaceutical Excipients or additives. This statement was particularly surprising in view of the tendency of the cytokines, e.g. IL-4R, to both decomposition as well as aggregation. In a specific embodiment of the invention the inhalable composition is a "neat" dry powder formulation another embodiment is the dry powder formulation without special excipients and Additives, e.g. Penetration enhancer, before.

In another embodiment can the compositions of the invention IL-4R together with one or more pharmaceutically acceptable Excipients or additives that are respiratory and pulmonary Administration are included. Such excipients, if present, are generally in the powder composition in amounts in the range of about 0.01% to about 99% by weight, preferably from about 0.1% to about 95%, stronger preferably from about 0.5% to about 80%, even more preferably from about 1% to about 50-60%. examples for Excipient-containing respirable IL-4R compositions are disclosed in U.S. Pat Example 1 described. Interestingly, influenced in the in Exemplary compositions described in the Examples Presence or absence of one or more excipients the chemical or physical stability the spray-dried, according to the invention Powder not essential, neither during still producing the storage.

however Some of the preferred excipients will serve one or more the following: the aerosol properties of the composition, their chemical stability, their physical stability and / or storage stability. Preferred excipients can also serve to provide more efficient and reproducible delivery of the To provide IL-4R by a dry powder inhaler and additionally the handling properties of the IL-4R powder composition (e.g., flowability and consistency) improve to facilitate production and powder filling.

In particular, the excipient materials can often serve to enhance the physical and chemical stability of the inhalable IL-4R powder composition or the active ingredients contained therein. For example, the excipient can minimize residual moisture content and stop moisture absorption and / or improve particle size, degree of aggregation, surface properties (ie, rugacity), ease of inhalation, and targeting of the resulting particles to the lung. The excipient (s) may also be used merely as fillers to reduce drug concentration in the tro serve powder composition.

In Pharmaceutical excipients useful in the present composition and additives close a, but are not limited to proteins (i.e., large molecules composed of one or more amino acid chains in a specific Order), oligopeptides (i.e., short, linked via peptide bonds Amino acid chains) Peptides (i.e., a class of molecules that hydrolyze into amino acids), Amino acids, Lipids (i.e., fatty, waxy or oily compounds, typically insoluble in water but in organic solvents soluble, the carbon is hydrogen and, to a lesser extent, oxygen contain), polymers (i.e., large molecules formed by the combination of many similar, smaller molecules) and Carbohydrates (e.g., sugars, including monosaccharides, di-, Tri-, tetra- and oligosaccharides; derivatized sugars, e.g. Alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present individually or together. Suitable excipients shut down those described in International Publication No. WO 96/32096, Inhale Therapeutic Systems, Inc. assigned.

preferred Close excipients Sugar alcohols, lipids, DPPC, DSPC, calcium / magnesium and hydrophobic Excipients, e.g. hydrophobic amino acids and hydrophobic sugars, one. Particularly preferred excipients include zinc salts, leucine, citrate and sugars, e.g. Raffinose, a.

For particulate formulations For example, preferred excipients are those which have glass transition temperatures (Tg) above about 35 ° C, preferably over about 45 ° C, stronger preferably over about 55 ° C.

typical Polypeptide and protein excipients include serum albumin, e.g. Human serum albumin (HSA), recombinant human albumin (rHA), gelatin, Casein, hemoglobin and the like. Particularly preferred are the dispersibility increasing Polypeptides, e.g. HSA, as in the international publication, No. WO 96/32096 assigned to Inhale Therapeutic Systems, Inc.

Representative Amino acid / polypeptide components, that can also work in terms of buffering capacity, include alanine, Glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, Leucine, isoleucine, valine, methionine, phenylalanine, aspartame, tyrosine, Tryptophan and the like. Preference is given to amino acids and Peptides that serve as dispersants. Amino acids that fall into this category include hydrophobic amino acids, such as e.g. Leucine (leu), valine (val), isoleucine (isoleu), tryptophan (try), Alanine (ala), methionine (met), phenylalanine (phe), tyrosine (tyr), Histidine (his) and proline (pro). A particularly preferred amino acid is the amino acid Leucine. Leucine closes, when used in the formulations described herein, D-leucine, L-leucine and racemic leucine. Dispersibility-enhancing peptides for use in the invention include dimers, trimers, tetramers and pentamers consisting of hydrophobic amino acid components, e.g. those described above, are composed. Examples shut down a di-leucine, di-valine, Di-isoleucine, di-tryptophan, di-alanine and the like, tri-leucine, Tri-valine, tri-isoleucine, Tri-tryptophan, etc .; mixed di- and tripeptides, e.g. leu-val, isoleu-leu, try-ala, leu-try, etc. and leu-val-leu, val-isoleu-try, ala-leu-val and the like, and homo-tetramers or Pentamers, e.g. Tetra-alanine and penta-alanine. Especially preferred oligopeptidic excipients are dimers and trimers consisting of two or more leucine residues are composed, as in the international Patent Application PCT / US00 / 09785 of Inhale Therapeutic Systems Inc. titled "Dry Powder Compositions Having Improved Dispersibity. "Among these are Dileucin and trileucine particularly preferred.

One another preferred feature of an excipient for use in The invention is the surface activity. surfactants Excipients, which may also serve as dispersants, such as e.g. hydrophobic amino acids (e.g., leu, val, isoleu, phe, etc.), di- and tripeptides, polyamino acids (e.g. polyglutamic) and proteins (e.g., HSA, rHA, hemoglobin, Gelatin) are particularly preferred because of their surface-active The nature of these excipients tend to be on the surface of the Concentrate particles of the respirable IL-4R composition, which makes the resulting particles highly dispersible in nature power. Other typical surface active Agents described in the respirable IL-4R compositions described herein can be included shut down a, but are not limited on polysorbate, lecithin, oleic acid, benzalkonium chloride and sorbitan esters.

to For example, carbohydrate excipients useful in the invention include Monosaccharides, e.g. Fructose, maltose, galactose, glucose, d-mannose, sorbose and the like; Disaccharides, e.g. raffinose, Meliticose, maltodextrins, dextrans, starches and the like; and alditols, such as. Mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), Myoinositol and the like.

The respirable IL-4R compositions may also include a buffer or include a means for adjusting the pH; typically the buffer is one made from an organic acid or base Salt. Representative Close the buffer Salts with organic acids, such as. Salts of citric acid, ascorbic acid, gluconic, Carbonic acid, Tartaric acid, Succinic acid, acetic acid or phthalic acid, Tris, Tromethamine hydrochloride or a phosphate buffer.

In addition, can the inhalable according to the invention IL-4R composition polymeric excipients / additives, such as e.g. Polyvinylpyrrolidones, derivatized celluloses, e.g. hydroxypropyl methylcellulose, Ficcole (a polymeric sugar), hydroxyethyl starch, dextrates (e.g., cyclodextrins, such as. 2-hydroxypropyl-β-cyclodextrin and sulfobutyl ether-β-cyclodextrin), polyethylene glycols, Pectin, flavors, salts (e.g., sodium chloride), antimicrobial Active ingredients, sweeteners, Antioxidants, antistatic agents, surfactants (e.g., polysorbates, such as. "TWEEN 20 "and" TWEEN 80 "), lecithin, oleic acid, benzalkonium chloride, Sorbitan esters, lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol) and chelating agents (e.g., EDTA). For compositions, which contain a polymeric constituent, the polymer is typically; to a limited extent in the composition is present, i. in shares of less than about 10% by weight. Preferred compositions according to the invention are those in which the IL-4R is preferably encapsulated non-liposomally or polymerically is or not-coated (i.e., without a delineated coating layer). Preferred IL-4R compositions, such as. those exemplified here are fast-acting Formulations, i. rather for fast applications as for Applications with extended Release designed.

Other for use in the inhalable IL-4R compositions of the invention suitable pharmaceutical excipients and / or additives in "Remington: the Science & Practice of Pharmacy ", 19. Ed., Williams & Williams, (1995), in Physician's Desk Reference, 52nd ed., Medical Economics, Montvale, NJ (1998) and in "The Handbook of Pharmaceutical Excipients ", 3. Edition, A.H. Kibbe, eds., American Pharmaceutical Association, Pharmaceutical Press, 2000.

According to the invention For example, the respirable IL-4R powder composition may be a dry powder be the dry powder crystalline, an amorphous glass or is a mixture of both forms. For formulations containing a surface active Contains agents, the surface-active material (either in crystalline or amorphous form) typically on the surface of the Particles in a higher Concentration may be present than in the bulk powder.

D. Preparation of the respirable IL-4R compositions

inhalable IL-4R powder compositions, e.g. Dry powder formulations are preferably by spray drying produced. The spray drying For example, the formulations are carried out as generally in the "spray-drying Handbook ", 5th ed., K. Masters, John Wiley & Sons, Inc., NY, NY (1991), in Platz, R., et al., International Patent Publications, Nos. WO 97/41833 (1997) and WO 96/32149 (1996).

Around an IL-4R solution for spray drying IL-4R (and any other excipients) becomes general dissolved in water, which optionally contains a physiologically compatible buffer. The pH range the solution is generally between about 3 and 10, with near neutral pHs are preferred because such pHs maintain the physiological compatibility of the powder after dissolution of the Powder within the lungs can support. The aqueous formulation may optionally additional water-miscible solvents, such as. Acetone, alcohols and the like. Representative Alcohols are lower alcohols, e.g. Methanol, ethanol, propanol, Isopropanol and the like. The solutions are generally IL-4R, solved at a concentration of 0.01% (w / v) to about 20% (w / v), preferably from 0.1% to 10% (w / v), more preferably 1% to 3% (weight / volume). In another embodiment can the components of the IL-4R formulation using an organic solvent or co-solvent system spray dried where one or more solvents, such as. Acetone, alcohols (e.g., methanol and ethanol), ethers, aldehydes, Hydrocarbons, ketones and polar aprotic solvents, be used.

The IL-4R containing solutions are then spray dried in a conventional spray dryer, such as those available from commercial suppliers, such as Niro A / S (Denmark), Buchi (Switzerland) and the like, resulting in a dispersible, respirable IL- 4R composition, preferably in the form of an inhalable dry powder. The optimum conditions for spray-drying the drug solutions will vary depending on the formulation ingredients and are generally determined experimentally. The gas used to spray-dry the material is typically air, although inert gases such as nitrogen or argon are also suitable. Moreover, both the inlet and outlet temperatures of the gas used to dry the sprayed material are such that they do not cause decomposition of the IL-4R in the sprayed material. Such temperatures are typically determined experimentally, although generally the inlet temperature will range from about 50 ° C to about 200 ° C, while the outlet temperature will be in the range of about 30 ° C to about 150 ° C.

The Preparation of the respirable, dry IL-4R powder compositions under a variety of different process parameters of spray drying is described in Examples 1 and 2. Surprisingly, the lead Shear forces, by the atomization the solution while caused by spray drying not to hydrolysis or aggregation of IL-4R. Like here described, can highly dispersible dry powders that are good physical and chemical stability and have good aerodynamic properties, reproducible and manufactured under many different process conditions.

In some cases may be desirable the respirable IL-4R powdered coconut powder formulation in a form which has improved handling / processing properties, e.g. reduced static, better flowability, low caking and the like, by making compositions, that of aggregates of fine particles, that is, aggregates or agglomerates of the respirable IL-4R described above are. Dry powder particles, the aggregates for the pulmonary Easily shattered again to fine powder ingredients be such. in Johnson, K., et al. U.S. Patent No. 5,654,007, 1997, described. In another embodiment, the inhalable IL-4R powder are made by agglomeration of the powder ingredients, sieving the Materials to obtain the agglomerates, spheronize to a more spherical To provide agglomerate, and classify to produce a product with uniform To get size such as. in Ahlneck, C., et al., PCT International Publication No. WO 95/09616 (1995).

The respirable IL-4R dry powders are used during manufacture, processing and storage, preferably under dry conditions (i.e., relatively low Moisture). Independently Of the drying method used, the method is preferably lead to respirable, highly dispersible compositions which essentially composed of amorphous IL-4R particles.

E. Properties of the inhalable IL-4R powder compositions

Certain physical properties of the spray-dried IL-4R powder compositions preferred to increase the efficiency of the aerosolized delivery of such To maximize compositions to the lungs.

The respirable IL-4R powder compositions are composed of particles, which are effective to invade the lungs. The passage of Particles in pulmonary physiology is an important consideration of the present invention. For this purpose, the particles of the invention a mass median diameter (MMD) of less than about 10 microns, preferably less than 7.5 μm and stronger preferably less than 5 μm, up and are usually in the range of 0.1 μm up to 5 μm In diameter. Preferred compositions are composed of particles, which have an MMD of about 0.5 to 3.5 microns. Examples of inhalable IL-4R powder compositions at varying concentrations Active ingredient (s) and / or excipient are described in Example 1. The respirable IL-4R powder compositions may also contain non-respirable carrier particles, such as. Lactose, containing the non-respirable particles typically larger as about 40 microns. In a preferred embodiment the dry powder is non-liposomal or non-lipid-containing.

The inhalable, according to the invention IL-4R powder compositions are further characterized by an aerosol particle size distribution less than about 10 μm mass median aerodynamic diameter (MMAD), preferably less than 5.0 μm and stronger preferably less than 3.5 μm, characterized. The massemedianen aerodynamic diameter of the Powders are characteristically in the range of about 0.5-10 μm, preferably of about 0.5-5, μm MMAD, more preferred from about 1.0-4.0 μm MMAD and even stronger preferably from about 1.5 to 3.5 μm, lie.

The respirable IL-4R powder compositions, especially the respirable dry powder compositions, generally have a moisture content below about 10% by weight, usually below about 5% by weight and preferably below about 3% by weight. Such low moisture solids tend to show greater stability during packaging and storage.

The dry powders preferably have a bulk density in the range of about 0.1-10 g / cm ^3, preferably from about 0.25-4 g / cm ^3, more preferably from about 0.5-2 g / cc, and most preferably from about 0.7-1.4 g / cm ³ , on.

The delivered dose (ED) of these powders is greater than 30% and usually greater than 40%. Stronger Preferably, the ED of the powders of the invention is greater than 50% and is often larger than 55%.

One additional Measure to Characterization of the total aerosol performance of a dry powder is the fine particle dose (FPD) or fine particle fraction (FPF), the mass percentage of the powder, which is an aerodynamic Diameter of less than 3.3 microns describes. Dry powder having an FPF value greater than 40%, more preferably greater than 50%, even stronger preferably greater than 60%, are especially good for suitable for pulmonary delivery. Powder that is at least fifty percent contain aerosol particles ranging in size from 0.5 to 3.5 μm, are extremely effective at delivery in aerosolized form of the lung areas, including the alveoli.

The according to the invention, spray-dried, respirable IL-4R powder compositions are still thereby characterized in that they have a substantially unchanged Monomer content opposite that of her solution or suspension before spray drying. In other words triggers the spray-drying process no formation of dimers or other aggregates, causing the Monomer percentage in the composition would be affected. The is called, the change in the monomer content between spray-dried Powder and solution or suspension before spray drying is in the Essentially unchanged, "for example, the percentage the monomer content of the spray-dried Powder opposite that of the solution or suspension before spray drying preferably not more than about 15%, more preferably not more than about 10%, more preferred not more than about 7%, even stronger preferably about 5% or less, as represented by the representative, exemplified in the examples, IL-4 powder.

The according to the invention, spray-dried, respirable IL-4R powder compositions are "shelf stable", i.e. characterized by minimal formation of insoluble Aggregates and / or a minimal decrease in the monomer content at Storage over extended periods at extreme temperatures ("temperature stable") and humidities ("Moisture-stable"), for example experience the spray-dried, respirable IL-4R powder compositions minimal aggregate formation and minimal decrease in monomer content after storage over one Period (e.g., two weeks or more) at a temperature in the range from about 2 ° C up to about 50 ° C, preferably about 25 ° C, and / or a relative humidity in the range of 0% to about 75%, preferably about 33% RH. Specifically, the stored inventive spray-dried, respirable IL-4R powder compositions, preferably less as about 15% insoluble Aggregates (opposite the solutions or suspensions before spray-drying), more preferred less than about 10% insoluble Aggregates, stronger preferably less than about 7% insoluble aggregates, even more preferred about 5% or less insoluble Aggregates. In another embodiment experience the stored inventive, spray-dried, respirable IL-4R powder compositions preferably a decrease in monomer content, which is not bigger than about 20%, preferably no greater than about 10%, more preferably not bigger than about 7%, even stronger preferably about 5% or less.

It Importantly, the differences between inhalable powder-based Formulations and nebulised formulations. In spite of the fact that nebulous formulations of some so far considered to be "inhalable", that they are inhaled through the mouth and into the lungs, they are not "inhalable" as defined here. For example, you can nebulised formulations typically do not deepen the tissues Achieve lung area and through the epithelial cells in the Blood circulation are absorbed. Moreover, these are cloudy formulations solution-based, i.e. be in solution instead of being given in solid form.

Representative respirable IL-4R powder compositions for pulmonary delivery in Examples 1-5 provided.

F. Pulmonary administration the respirable IL-4R powder compositions

The respirable IL-4R powder compositions, especially the dry described herein Powder compositions are preferably delivered using a suitable dry powder inhaler (DPI), ie, an inhaler that uses the patient's inhaled breath as a vehicle to deliver the previously dispersed (by passive or active agent) dry powder into the lungs. Preferred are dry powder inhalers manufactured by Inhale Therapeutic Systems as described in Patton, JS, et al., U.S. Patent No. 5,458,135 (1995); Smith, A., et al., U.S. Patent No. 5,740,794, (1998); Smith, A., et al., U.S. Patent No. 5,785,049 (1998) and International Patent Application PCT 00/18084.

at Administration using a device of this type is the inhalable IL-4R powder composition contained in a receptacle, which a pierceable Cover or another access surface, preferably a blister pack or cartridge, wherein the receptacle is a single Dosage unit or multiple dosage units may contain. A big Number of wells are conveniently measured with Doses of dry powder medicine filled, as in Parks, D.J. et al., International Patent Publication WO 97/41031 (1997).

Also for delivery of the inhalable IL-4R powder formulations described herein suitable are dry powder inhalers of the type for example in Cocozza, S., U.S. Patent No. 3,906,950 (1974) and Cocozza, S., U.S. Patent No. 4,013,075, (1977), with a prior metered dose of dry powder for delivery to an individual contained within a hard gelatin capsule.

Other dry powder dispersing devices for the pulmonary administration of dry powders include those described, for example, in Newell, RE et al., European Pat. EP 129 985 (1988); in Hodson, PD et al., European patent no. EP 472 598 (1996); in Cocozza, S., et al., European patent no. EP 467172 (1994) and in Lloyd, LJ et al., U.S. Patent No. 5,522,385 (1996). Also suitable for delivery of the IL-4R powder compositions of the present invention are inhalation devices, such as the Astra-Draco "TURBUHALER." This type of device is disclosed in Virtanen, R., U.S. Patent No. 4,668,218, in Wetterlin, K. et al No. 4,667,668 (1987); and in Wetterlin K., et al., U.S. Patent No. 4,805,811 (1989).) Also suitable are devices which employ the use of a piston to draw air for either entrainment the powdered medicament, releasing the medicament from a carrier screen by passing air through the screen, or mixing the air with the powder medicament in a mixing chamber, with subsequent introduction of the powder into the patient through the mouthpiece of the device, such as in Mulhauser, P. , et al., U.S. Patent No. 5,388,572 (1997).

The inhalable IL-4R powder compositions may also be used of a pressurized metered dose inhaler (MDI) be that a solution or suspension of the drug in a pharmaceutically inert liquid Propellant, e.g. a chlorofluorohydrocarbon or a fluorinated one Hydrocarbon contains, as in Laube, et al., U.S. Patent No. 5,320,094 (1994) and in Rubsamen, R.M. et al., U.S. Patent No. 5,672,581 (1994). Before use are the respirable IL-4R powder compositions generally in a receptacle under Environmental conditions are stored and are preferably at temperatures at or below about 25 ° C and relative humidities (RF) in the range of about 30 to 60% stored. Stronger preferred relative humidity conditions, e.g. less than about 30%, can by introducing a desiccant into the secondary packaging the dosage form can be achieved. The inhalable invention Dry powders are not only characterized by good aerosol performance, but also by good stability.

at Aerosolization for direct delivery in the lungs will be the here described IL-4R powder compositions good lung bioavailabilities demonstrate.

G. benefit

The inhalable IL-4R powder compositions of the invention are particularly effective in the treatment of allergic diseases and disorders, such as asthma, atopy, atopic dermatitis and other conditions associated with high serum IgE and IgG ₁ levels in pulmonary use.

The respirable IL-4R powder compositions may also be used to treat or prevent allergic, viral, parasitic, and bacterial diseases and mold infectious diseases, particularly when administered together with gamma interferon. (See European patent no. EP 585 681 (1994)).

The invention powder compositions penetrate into the airways of the lungs when inhaled, enter the bloodstream and achieve effective systemic delivery. Moreover, the pulmonary doses of the IL-4R powder are typically much lower than those given orally because of the loss associated with digestion and disintegration for oral dosage forms.

The inhalable, according to the invention IL-4R powder compositions find use as substitutes or additives for common ones Asthma therapies.

The inhalable IL-4R powder compositions find particular application in the physiological regulation of serum levels of IL-4 and related immunoglobulins (eg, IgE and IgG ₁ ).

The respirable IL-4R powder compositions continue to be used as an amplifier the biological activity of the cytokine IL-4, thereby leaving room for the reduction of the required Cytokine dosing and minimizing the associated negative Side effects (See U.S. Pat No. 6,063,371).

The The following examples illustrate the scope of the present invention Invention, but should not limit it in any case.

EXAMPLES

materials and methods

The The following materials were used in the following examples:

materials

• L-leucine (Aldrich, St. Louis, MO)
• hydrochloric acid (J. T. Baker, Phillipsburg, N.J.)
• Sodium Hydroxide 0.1N Titration Solution (J. T. Baker, Phillipsburg, N.J.)
• Ethanol, 200 proof (USP / NF, Spectrum Chemical Mfg. Corp., New Brunswick, N.J.)
• Methanol (HPLC-pure, EM Industries, Gibbstown, N.J.)
• Recombinant human IL-4R (rhuIL-4R) (Immunex Corporation, Seattle, WA)
• male Sprague-Dawley rats (Simonsen laboratories, Gilroy, CA)

Powder Analysis

IL-4R solutions: The following solutions with IL-4R were prepared. Solution A contained 22.6 mg / mL serum-free IL-4R in 5 mM NaH ₂ PO ₄ buffered to pH 7.0. Solution B contained 6.1 mg / mL serum-free IL-4R in distilled water. The solution concentrations are expressed as the mass of the peptide per unit volume. Values reported here are from amino acid analysis data; Values from the UV test were slightly higher (25.6 and 7.5 mg / mL, respectively).

Particle size. The particle size distribution The typical IL-4R powder was made with a liquid centrifuge settling process in a Horiba CAPA-700 particle size analyzer following the Dispersion of powders in SediSperse A-11 (Micrometrics, Norcross, GA).

Moisture content. The moisture content of the powder was using the titrimetric Karl Fischer titration using a Mitsubishi CA-06 Moisture Meter or measured by thermogravimetric analysis.

MMADs. The aerosol particle size distribution (MMAD) was measured using a cascade impactor (Graseby Andersen, Smyrna, GA) at a flow rate of 28L / min determined, ignoring the loss of powder at the inlet manifold has been.

Number of Votes Dose. Delivered doses were determined as described in the section "Definitions", below Use of an Inhale dry powder inhaler as in U.S. Patent No. 5,740,794 described, and a Gelman glass filter, 47 mm diameter.

Scanning Electron Microscopy (SEM). Particle morphology was determined using egg XL 30 ESEM manufactured by Philips Electron Optics (Eindhoven, The Netherlands).

Thermal analysis. Thermal analysis experiments were carried out using a modulated differential scanning calorimeter (mDSC model 2920), a dielectric analyzer (DEA 2970) and a thermogravimetric analyzer (TGA Model 2950, manufactured by TA Instruments (New Castle, DE). Hot Stage Microscopy was using a Nikon Optiphot 2-pin optical microscope (Nikon Inc., Torrance, CA), a Hamatsu camera and a C2400 Controller (Hamatsu Photonics, Herrsching, Germany), a Mettler-Toledo FP90 central processor (Mettler-Toledo, Columbus, OH) and a FP8902H hotplate accessory (Mettler, Toledo, OH).

Dry Characterization: SE-HPLC experiments were performed on a Waters HPLC system Alliance Model 2690 (Alliance HPLC Systems, Millford, MA) with Millennium V-Software (Waters). Chromatography columns were obtained from Phenomenex (Torrance, CA). UV tests were done on one Hitachi U-3000 dual-beam spectrophotometer (Hitachi Instruments Inc., San Jose, CA). SDS-PAGE experiments were performed on a Novex Xcell electrophoresis unit (Novex, San Diego, CA).

example 1

Preparation of IL-4R dry powders

Shelf stable spray-dried Powder of interleukin receptor protein, IL-4R, the outstanding Have aerosol properties and continue to provide excellent chemical and physical stability were identified produced. The powders were both present and absent produced by excipients; the excipients used were derived from many different representative ones chemical classes (e.g., salts with organic acids, amino acids, metal cations). The IL-4R powders are stable on long-term storage and are resistant to extreme conditions Temperature and humidity conditions.

Representative IL-4R powders were prepared according to the following Protocols produced.

Example 1 (A) - undiluted formulation with IL-4R

Dry powder compositions with IL-4R were dissolved in deionized water without added excipients for the spray drying formulated. 600-700 mg batches of undiluted Formulations with IL-4R were made by spray-drying a suitable Volume of solution A made. The final concentration of the phosphate buffer in the solution Spray drying was 1.9 mM.

Example 1 (B) - zinc chloride-containing Formulation with IL-4R

Dry powder compositions with IL-4R were formulated in deionized water with zinc chloride for spray drying. 600-700 mg batches of 5.4: 1 ZnCl ₂ : IL-4R formulations were prepared by pouring 19.53 mL of IL-4R solution A and 0.456 mL of a 19.37 mg / mL solution with ZnCl ₂ into a 50 mL volumetric flask and adjust the final volume to 50 mL by adding deionized water. The final concentration of the phosphate buffer was 1.9 mM.

Example 1 (C) - citrate-containing Formulation with IL-4R

Dry powder compositions with IL-4R were dissolved in deionized water containing a citrate salt, for the spray drying formulated. 600-700 mg batches of citrate: IL-4R formulations were prepared by combining approximately 12 mL of solution A and 200 mg citrate in solution at pH 7.5 and adjusted to a final volume of 50 mL Add deionized water. The final concentration of the phosphate buffer was 1.2 mM.

Example 1 (D) - Leucine-containing Formulation with IL-4R

Dry powder compositions with IL-4R in deionized water containing leucine for spray drying formulated. 600-700 mg batches of leucine: IL-4R formulations were prepared by combining approximately 12 mL of solution A and 200 mg leucine and adjust to a final volume of 50 mL Add deionized water. The final concentration of the phosphate buffer was 1.2 mM and the pH was 7.5.

The relative amounts of ingredients (w / w) in the formulations 1 (A) -1 (D) are summarized in Table 1 below.

Table 1 Formulations with IL-4R

additional contemplated powder formulations include IL-4R formulations, both undiluted as also containing excipients, which are made using a citrate buffered IL-4R solution or an IL-4R solution water-based (no buffer) were made. Preferred IL-4R powders according to the invention comprise in addition to IL-4R, one or more of the following excipients: trileucine, Raffinose, mannitol, sucrose, F-68, divalent metal cations, such as. Magnesium, calcium and the like, glucophosphates, zinc salts, Trehalose, glycine and histidine. Specific formulations may be of 10-40 Wt% trileucine or 1 wt% F-68 or 10 wt% citrate or 3: 1 to 10: 1 by weight cationic divalent metal cation: IL-4R or 10-30 Wt .-% sucrose or 5-50 Wt% trehalose or any combination of the above. additional Illustrative IL-4R formulations include those that are both Citrate as well as leucine (e.g., a formulation with a citrate: leucine: IL-4R ratio of 15:15:70) or raffinose (e.g., formulations containing 5% -50% raffinose include).

The The above IL-4R formulations were prepared using a Büchi Mini Spray Dryer in the Laboratory scale (Büchi Labortechnik, Ag., Meierseggstrasse, Switzerland), equipped with a modified cyclone, a spray nozzle and a powder trap, spray dried. The atomizer of the Büchi spray dryer was operated with clean, dry air. The flow rate the liquid in the Büchi was 5 mL / min. The inlet temperature was adjusted to the Target particle size and morphology to reach (80 ° C up to 150 ° C). The outlet temperature was in the range of about 30 ° C to 100 ° C. The yields were greater than 75% and ranged from 78-91% for the IL-4R Formulation Batches 1 (A) -1 (D). The IL-4R powders (3 ± 0.15 mg) were placed in a glove box with a relative humidity of less than 5% convicted and placed in unit dose forms (blister packs, BPs), for use in a dry powder inhaler, as in U.S. Patent No. 5,740,794. The review and characterization of the thus-obtained solids will be described below described.

Example 2

Stability of IL-4R powder formulations

Of the The purpose of the examinations of Examples 2-5 was to study the aerosol performance, physical and chemical stability of those described in Example 1 representative IL-4R dry powder formulations to rate. Aerosol, thermal, physical and chemical tests were carried out on the powders as prepared initially. Thermo and physical and chemical analyzes were also done after 2 weeks Storage at four different temperatures (2-8 ° C, 25 ° C, 40 ° C and 50 ° C) and 3 relative humidities at 25 ° C (0.33 and 75%). The stability protocol is set out below:

The Powders were filled in foil / PVC blister packs and were only initial on the delivered dose, particle size distributions and thermal analyzes tested. The chemical characterization and SEM analyzes were on bulk aerosol drug powders (i.e., not contained in blister packs) initially and after 2 weeks executed. All powders were in moisture-controlled gloves boxes handled with a relative humidity of less than 5%.

• ¹ Test ausgeführt unter Verwendung von Blisterpackungen
• ² Test ausgeführt unter Verwendung von Schüttpulver
• ³ Schüttaerosolpulver, gelagert in verschlossenen und mit Parafilm umwickelten Borsilikatglas-Gläschen in einer versiegelten Folienumverpackung
• ⁴ Schüttaerosolpulver, gelagert in Borsilikatglas-Gläschen mit offenem Deckel

Tabelle 3: Wässrige Verdünnungen aus der Vorratslösung

• ⁵ Lösung, verdünnt mit Wasser aus der IL-4R-Lösung A
• ⁶ 1 mL einer 1 mg/mL-IL-4R-Lösung, gelagert in verschlossenen und mit Parafilm umwickelten Borsilikatglas-Gläschen in einer versiegelten Folienumverpackung

The bulk powder was weighed in a glove box in borosilicate glass vials. For stability pro At 0% relative humidity (RH), the vials were sealed, placed in a film overwrap bag containing a desiccant, and sealed in temperature chambers prior to storage. For moisture-controlled stability samples, the vials were left open and stored in desiccators at 25 ° C. After 2 weeks samples were taken and analyzed by UV, SDS-PAGE, SE-HPLC and SEM. Table 2: Summary of the stability protocol for spray dried IL-4R bulk powders and IL-4R stock solutions

• ¹ test performed using blister packs
• ² Test carried out using bulk powder
• ³ Bulk aerosol powder stored in sealed and parafilm-wrapped borosilicate glass vials in a sealed foil outer packaging
• ⁴ bulk aerosol powder stored in borosilicate glass jars with open lid

Table 3: Aqueous dilutions from the stock solution

• ⁵ solution diluted with water from IL-4R solution A
• ⁶ 1 mL of a 1 mg / mL IL-4R solution, stored in sealed and parafilm-wrapped borosilicate glass vials in a sealed foil overwrap

Example 3

Aerosol performance of IL-4R powder formulations

aerosol tests were executed using a dry powder inhaler as described in U.S. Pat. No. 5,740,794, assigned to Inhale Therapeutic Systems Inc. All filled Blister packs were used for aerosol testing before use Drying chamber stored.

Example 3 (A): Delivered dose

The delivered dose was measured by placing the aerosol on an in a holder over the mouthpiece the chamber of the device placed glass fiber filter was caught. By the percent delivered To measure dose (ED%), a blister pack was used a dry powder inhaler as described above as an aerosol dispersed. The powder sample was on a before weighed glass fiber filter (Gelman, 47 mm diameter). The aerosol cloud was caught on the filter by the chamber, by for 2.5-3.5 Seconds with an air flow speed of 30 ± 0.5 L / min was pulled. An automatic timer controlled the duration of the train. The sampling pattern simulates slow, deep inhalation of the patient.

The ED% were calculated by taking the mass of the collected on the filter Powder through the mass of the powder in the blister pack (actual Mass) was divided. The results are reported in Table 4. Each reported result was the mean and standard deviation out of 10 measurements.

Example 3 (B): Mass Median aerodynamic diameter (MMAD) and geometric standard deviations (GSD)

Of the massemediane aerodynamic diameter and particle size distribution of the aerosol were measured using an 8-step (9.0, 5.8, 4.7, 3.3, 2.1, 1.1, 0.7 and 0.4 μm Separation grain sizes) of Andersen Cascade Impactors receive. Each Andersen measurement was obtained by adding 10 blister packs with 3 mg filling weight in a dry powder inhaler were dispersed while for 2.5 Seconds at 28.3 ± 0.5 L / min a vacuum was pulled. An automatic timer controlled the duration of the train. From these data became a massemedianer aerodynamic Diameter calculated. The results are set forth in Table 4.

The geometric standard deviations (GSDs) were graphically from the Andersen MMAD data calculated. The diameter of the separator plate was considered a function the cumulative percent screen diarrhea on a probability scale applied. The GSD was divided as the diameter at 85% by the Diameter taken at 50%. The values obtained are shown in Table 4 listed.

Example 3 (C): Fine particle fraction (FPF)

• * RSD für n = 3
• ** Die Werte sind der Mittelwert und RSD, in Klammern, von 10 Messungen.

The FPF _{% <3.3μm} was obtained using the Andersen Cascade Impactor stages F and 3 in short order. Each FPF measurement was obtained by dispersing two 3-mg blister packs in a dry powder inhaler while drawing a vacuum at 28.3 L / min for 2.5 seconds. The results are set forth in Table 4. Table 4 Initial aerosol test results for IL-4R formulations (3 mg fill weight)

• * RSD for n = 3
• ** The values are the mean and RSD, in brackets, of 10 measurements.

The Aerosol performance of the IL-4R powder formulations was quite good, all had ED values of substantially 60% or greater and MMAD values of 4 μm or less, with at least 34% of the particles being MMADs of less than 3.3 μm exhibited.

The GSD values were all less than 2 μm, except for the leucine formulation. It is believed that this is an artifact due to the observed, from the Andersen AI generated binodal particle size distribution was.

Example 4

Characterization of the IL-4R powder formulations in solid state

Example 4 (A) - Scanning Electron Microscopy

scanning electron Microscopy was used to provide initial morphological information about the spray-dried To get powder and change the morphology when stored under varying temperature and To assess moisture conditions.

The images were taken with a Philips XL30 ESEM operated in high vacuum mode using an Everhart-Thornley detector to capture secondary electrons for image composition. The acceleration voltages were 3 to 10 kV, using a LaB ₆ source. The working distances were in the range of 30 to 10 microns.

All Powder except leucine: IL-4R powder, Formulation 1 (D), showed no noticeable change morphology after 2 weeks of storage in the stability protocol of Example 2 (Table 2) described temperature and RF condition. Some of the leucine: IL-4R powders showed morphological changes at RF values of 33 to 75% RH, and at temperatures of 40 to 50 ° C, characterized by band-like Growth due to crystallization of the amorphous leucine can.

at visual exam with SEM can the particles show a wrinkled, raisin-like morphology. Rugose particles (i.e., particles with deep wrinkles, which are essentially more rough than smooth) are most preferred.

The effects of temperature and relative humidity were for representative IL-4R formulations determined. Of the illustrative IL-4R powders prepared, the spray-dried leucine powder appeared to be the least morphologically stable based on temperature and RF induced changes in morphology. No significant morphological changes were noted on any of the other powders when exposed to identical storage conditions.

Example 4 (B) - Thermal analysis

differential (DSC): DSC profiles were generated by placing a sample in a hermetic sealed dish in a TA Instruments DSC. The Sample weights were of the order of magnitude from 3.5-4.5 mg. The DSC heating rate was 10 ° C / min with helium as the purge gas. The Temperature was initially at 70 ° C elevated, with 10 ° C / min cooled to -30 ° C and with 10 ° C / min back to 220 ° C heated to the heat-history to remove instead of using the modulation mode. All powders showed a big one endothermic signal at approximate 198 ° C. The zinc-containing IL-4R formulation, Formulation 1 (B), showed an additional endothermic signal at about 180 ° C. The citrate-containing IL-4R formulation, Formulation 1 (C), showed an additional endothermic signal at about 172 ° C. The leucine-containing IL-4R formulation, Formulation 1 (D), showed an additional endothermic signal at about 186 ° C. Using this method, no glass transition was made to the powders (Tg) noticed.

Consequently show the spray-dried according to the invention Powder glass transition temperatures, the much higher are room temperature, a preferred property of dry powder formulations, especially for Long-term storage. Thus, in another embodiment, the representative ones IL-4R powder according to the invention marked by Tgs, the higher are as 100 ° C. Due to the high Tgs of the powder according to the invention, these can IL-4R powder at temperatures above Ambient temperature or 25 ° C be stored stable and can at 30 ° C, 35 ° C, 40 ° C, 45 ° C, 50 ° C, 55 ° C, 60 ° C, 65 ° C or higher (to about 100 ° C or even higher) for extended periods (e.g., one week, two weeks, one month, two months, three to six months, nine months, up to a year or more) stable be stored while their advantageous aerosol properties maintained (with storage essentially a minimal Drop in delivered dose of not more than about 15%, preferably not more than about 10%, and even more preferably not more than show about 5%, and essentially no change in MMAD, like by an increase in MMAD of not more than about 1 micron and preferably not more than about 0.75 microns, and more preferably not more than about 0.5 microns).

Dielectric Relaxation Spectrometry (DRS): Since the glass transition temperature (Tg) of the spray-dried IL-4R formulations could not be determined by DSC, DRS was performed. Two DRS experiments were performed on the zinc-containing IL-4R formulation, Formulation 1 (B), to better detect the glass transition temperature of a representative formulation. The first experiment carried out was a standard DRS analysis (TA Instruments Dielectric Analyzer (DEA 2970)) performed at 2 ° C / min. From 30 ° C to 150 ° C and again cooled to 30 ° C using frequencies of 1 , 10, 100, 10 ³ and 10 ⁴ and 10 ⁵ Hz was scanned. The second experiment carried out was a softening experiment, quite similar to the thermomechanical analyzer (TMA), performed at 2 ° C / min from 30 ° C to 250 ° C and with frequencies of 1, 10, 100, 10 ³ and 10 ⁴ and 10 ⁵ Hz was scanned. Both experiments were performed on the Zn: IL-4R powder per se (Formulation 1 (B)) and after drying overnight at 100 ° C (Formulation 1 (B) - (D)).

Out These investigations concluded that there is no glass transition below the decomposition temperature for the dried sample and no glass transition below 150 ° C for the undried sample gives. This indicates that the with DSC observed changes in the baseline below 100 ° C is not due to a glass transition event were due. Thus, the Tg of this formulation is greater than the decomposition temperature of the protein.

Thermomechanical Analysis TMA): The TMA experiments were carried out by monitoring the sample thickness during a DRS experiment. The same electrode configuration was used as in the first experiment, except that the gasket was removed. The piston pressure was set to 20N and the thickness of the sample and the temperature were manually recorded every few minutes from 30 ° C to 250 ° C. The onset of softening is at 224 ° C for both Formulation 1 (B) and Formulations 1 (B) - (D). The softening seen in the TMA experiments is due to decomposition and a possibility of concurrent glass transition. Since there is no other softening that occurs at lower temperatures, the 1Hz peaks from the standard DRS tests are due to some other mechanism, such as the onset of side chain movement or ionic conduction, and are not due to a glass transition (Seyler, et al. RJ, 1994, "Assignment of the Glass Transition", ASTM, 108-113) Glass transition occurs simultaneously with the decomposition, then in the sample with the loss factor peak below 1Hz, the glass transition may also be shifted to a lower temperature. Since the standard DEA test was performed only up to 150 ° C, it is clear that there is no glass transition below 150 ° C. The evaluation of the plots plotting the dielectric constant versus temperature confirmed the standard s-shaped profiles expected for this type of analysis.

thermogravimetric Analysis (TGA): The residual solvent content in the powder after spray drying was determined with TGA using a TA Instruments TGA. Nearly 3 mg of powder were placed in a hermetically sealed aluminum pan in a glove box with a relative humidity of less than 3% packed. The TGA was set to zero without the shell and the weight of the powder was recorded in the comment section. Just before the Analysis, the shell was pierced with a needle and into the Apparatus loaded. The scanning speed was 10 ° C / min of 25-175 ° C. The results will be shown in Table 5.

Table 5 Solvent content of the spray-dried IL-4R powders according to TGA

Hot Stage Microscopy (HSM): Hot stage microscopy was used from room temperature to 220 ° C a Nikon Optiphot 2-pin optical microscope, a Hamatsu camera and a C2400 controller, a Mettler Toledo FP90 central processor and FP8902H Hot Top Mixer carried out. Low to no change in the visual appearance of the powder was at the dry Preparations observed. The undiluted (NM1392-04) and the zinc (NM1392-05) formulations showed some particles that "burst open" or jumping of the particles at approximate 80 ° C. at all formulations rated in high temperature immersion oil Channels were formed at approximate 90 ° C and at 110 ° C outgassing was observed. It is believed that this outgassing due to loss of water from the powder. The extended time for this Outgassing is consistent with those observed with DSC down sloping profiles.

Example 5

chemical characterization of the IL-4R powder formulations

Several Techniques can can be used to analyze the samples of Example 1 to to determine the degree of aggregation and decomposition. Insoluble aggregates were determined by visual detection and UV spectrophotometry. Soluble aggregates were quantitated by size exclusion chromatography and qualitatively analyzed by SDS-PAGE.

Example 5 (A): Size exclusion high pressure liquid chromatography (SE-HPLC)

Soluble aggregates were quantitatively measured by SE-HPLC. The samples were taken to the Injection at 5 ° C stored. The chromatograms were extracted and processed at 220 nm.

Of the Percent monomer content of the formulated solutions before spray drying was mixed with the corresponding reconstituted aerosol drug powders compared. There were no significant changes in the percentage of monomer content between the formulated solutions and the reconstituted aerosol drug powders. The results are set out in Table 6 below.

Based on the results obtained from the powder stability temperature data at two weeks, the amount of monomer content increased as the storage temperature increased compared to the initial ones Data off. The largest change in the percentage of monomer from the initial value was at 50 ° C with a range of 2.1% in the neat formulation to 1.9% in both the zinc and citrate formulations. In the powder stability-humidity study after 2 weeks, the citrate formulations showed the largest drop to just 1.8% monomer from the initial value at the extreme RF of 75%. This was probably due to citrate crystallization. Thus, the IL-4R compositions of the present invention exhibit substantially no thermal degradation upon spray drying (as evidenced by the monomer content of the illustrative compositions) and exhibit a minimal decrease in monomer storage under many different illustrative temperature and humidity conditions. In contrast to other proteins which often tend to aggregate significantly in spray-drying (Maa, YF, et al., J. of Pharmaceutical Sciences, Vol. 87 (2), pp. 152-159 (1997)), the IL- 4R found that it is surprisingly resistant to and inaccessible to such conditions and forms spray-dried powders in which the protein shows negligible decomposition, even in the absence of commonly used stabilizing / protective excipients.

Two liquid 1 mg / mL samples stored at 25 and 50 ° C for 2 weeks were evaluated as controls. The liquid sample stored at 25 ° C had a 0.7% dimer content, expressed as a percentage of the monomer, but had over 17% low molecular weight species. At 50 ° C, the liquid had 2.3% dimer content, expressed as a percentage of monomer and 2.1% low molecular weight species. Samples were prepared and retested and the original results were confirmed. The drop in the percentage monomer content of the liquid IL-4R formulation at 50 ° C for 2 weeks is approximately 40% of the initial value (See Table 6 and Figs 1 . 2 and 3 ) while the powder formulations remained essentially unchanged. Thus, solution formulations with IL-4R are significantly more unstable (ie, prone to decomposition) than the corresponding powder formulations that have been stored under essentially the same conditions for a given period of time. Interestingly, the extreme temperature and shear conditions experienced by the IL-4R molecule in solution during spray-drying did not significantly decrease the monomer content or promote extensive chemical degradation of the molecule, such as observed for the stored liquid samples. The above data indicate the striking advantage of the powder formulations with IL-4R when stored versus the corresponding liquid formulations, based on both chemical and physical stability data.

Example 5 (B): Ultraviolet Spectroscopy (UV)

UV spectrophotometric analyzes were used to evaluate turbidity (ie aggregation / precipitation) in reconstituted samples. The measurements were carried out on a Hitachi U-3000 dual-beam spectrophotometer. The instrument parameters were set at a scanning speed of 300 nm / min; 1.0 nm gap width; and a scan was in the range of 450 nm to 200 nm. The samples were visually inspected for particulate matter. Insoluble aggregates were determined quantitatively by measuring the turbidity of the solution with UV. Linear regression was performed on the extinction values at 350, 375 and 400 nm to correct the scattering. The extinction corrected for light scattering at λ _max was extrapolated from the equation for the regression line. The percent insoluble aggregate is the percentage of absorbance corrected for light scatter divided by the uncorrected absorbance at λ _max , as shown in Eq. 1 shown.

The Samples were either reconstituted or analyzed prior to analysis Concentration of 0.1 mg IL-4R peptide / mL diluted with water.

Except for a sample, all solution samples before and after spray drying had no visible evidence of particulate matter or less than 5% calculated insoluble aggregates. The spray-dried neat formulation, Formulation 1 (A), was visibly clear under initial conditions but, when calculated according to Eq. 1 7% insoluble aggregates. Since at the 2-week stability time point this sample contained only 3% insoluble aggregates, it was concluded that the initial value was a mistake. All samples showed no visible signs of suspended matter or detectable insoluble aggregates after exposure for 2 weeks to specified temperature and humidity conditions. Fewer as 3.5% insoluble aggregates were calculated using Eq. 1 calculated for all batches. Table 6 only contains data obtained by SE-HPLC and contains no information on insoluble aggregates.

Table 6: Summary of aggregation for IL-4R formulations (SE-HPLC)

Example 5 (C): Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

Soluble aggregates and decomposition were qualitatively SDS-PAGE using a Novex Silver Xpress Coloring Kits measured. Prefabricated Novex 4-20% Tris-Glycine gels were developed on a Novex Xcell II Elektophoresis minicell. The samples were reconstituted or reconstituted with water Diluted concentration of 0.1 mg IL-4R peptide / mL. The solutions were under reducing and non-reducing conditions and with a load from 1-5 μg of protein applied per lane. The 5 μg protein loadings were designed to try detection in the 1 μg protein loading reinforce observable bands. The reduced samples were treated with 2-mercaptoethanol and at 100 ° C for 3 minutes heated. The gels were developed with 125 V, 25 mA / gel until the Gelfront reached the bottom (about 1.5 hours). In addition to the solution above Spray drying, The initial powders and the stability samples were a solution 1 mg IL-4R peptide / mL analyzed as a control. reducing and non-reducing Gels were for the liquid Initial formulation, developed at 25 ° C after 2 weeks and 40 ° C after 2 weeks.

It There were no changes in the gel profiles between the formulated solutions before spray-drying and the reconstituted aerosol drug powders. The monomer bands All samples and controls with IL-4R were on the gels higher Molecular weight (ann 50 kDa) than the reported values and appear wide and diffuse. This is most likely the glycosylation of the protein, which affects the migration of the IL-4R through the gel, attributed. There was another distinct bond that came to approximately 97 kDa ran, this was attributed to the dimer, which probably is the dimer form of the protein. Several low molecular weight bands were in the gel with 5 μg loading visible, which have not been identified.

As at the beginning powders, there were humidity tests at both temperatures compared to the control solution no visible changes in the stability gel profiles the bulk aerosol powder after 2 weeks. The at 25 ° C, 75% RF for two weeks incubated undiluted IL-4R sample was in the original Gel not detected, probably due to dilution error. With repeated analysis, the sample was equivalent to the other stability samples. The gels of the IL-4R solution samples showed a big one Degree of decomposition and aggregation compared to the spray-dried Powders.

In summary, IL-4R was based on the results obtained from the stability study in all four batches of bulk powder formulations (Formulations 1 (A) -1 (D)) for until for two weeks at 2-8 ° C, 25 ° C, 40 ° C and 50 ° C, next to 25 ° C at 0%, 33% and 75% RH for uncovered powder, observed to be chemically stable. With UV was for all batches are not insoluble Aggregation observed. The monomer content fell for all formulations in these Conditions by less than 2%, with the citrate formulation at Storage the biggest reduction in the monomer content showed.

Claims (22)

Spray-dried A powder composition comprising IL-4R and a pharmaceutical compatible excipients. A composition according to claim 1 in aerosolized Shape. A composition according to claim 1, wherein the excipient selected is made of carbohydrates, amino acids, Oligopeptides, peptides and proteins. A composition according to claim 3, wherein the carbohydrate is a sugar or sugar alcohol. Compositions according to claim 3, wherein the amino acid is a hydrophobic amino acid is. A composition according to claim 1, wherein the excipient selected is from citrate salts, leucine, raffinose, zinc salts and combinations from that. A composition according to claim 1, wherein the excipient is a buffer. A composition according to claim 1, wherein the excipient is a divalent metal cation. Composition according to claim 1, characterized by a given dose of at least 30%. Composition according to claim 9 by a delivered dose of at least 45%. Composition according to Claim 10 by a given dose of at least 60%. A composition according to claim 1 comprising particles with a mass median aerodynamic diameter (MMAD) of less than about 10 microns. A composition according to claim 1 comprising particles with a mass median aerodynamic diameter (MMAD) of less as about 5 microns. A composition according to claim 1 comprising particles with a mass median aerodynamic diameter (MMAD) of less as about 3.5 microns. A composition according to claim 1 comprising particles with a mass median aeordynamic diameter (MMAD) of about 0.1 to 3 microns. A composition according to claim 1 having a residual moisture content less than about 10% by weight. A composition according to claim 16, wherein the residual moisture content less than about 5% by weight. A composition according to claim 1, wherein the composition a bulk density from about 0.1 to 10 g / cc. A composition according to claim 1 in a unit dosage form. Process for the aerosolization of an IL-4R dry powder composition, the method comprising: (A) Providing an IL-4R composition according to claim 1 and (b) dispersing the composition into a gas stream to an aerosolized dry powder to form, which is suitable for inhalation. Method according to claim 20, wherein the dispersing by means of a dry powder inhaler is reached. Process for the preparation of a dry IL-4R powder composition, the method comprising: (A) Prepare a mixture or solution of IL-4R in a solvent and (b) spray-drying the mixture or solution, to the IL-4R powder according to claim 1 to get.